Studies with sera from ferrets infected with single influenza viruses and from people immunized with inactivated influenza vaccines performed by our laboratory confirmed that A/Wuhan/359/95 (W95 [H3N2]) was markedly different from previous vaccine strains. The data were used by the USPHS and WHO to recommend inclusion of a W95-like strain in inactivated influenza vaccines for the 1996-1997 influenza season. Our laboratory produced reference reagents for these strains to facilitate qualification and release of approximately 80 million doses of influenza vaccine for the United States. In addition, as a reference strain for production purposes, we developed and cloned a reassortant influenza virus with increased ability to replicate in eggs. The strain, designated RESVIR 9 combined the six internal genes of A/Puerto Rico/8/34 (PR8) [H1N1] with the hemagglutinin [HA] and neuraminidase [NA] genes of W95. The origins of the gene segments of RESVIR 9 were confirmed using our strategy combining polymerase chain reaction (PCR) and endonuclease restriction mapping. After confirmation, this strain was distributed to the WHO Influenza Centers, national laboratories and manufacturers. We discovered that the current method of quantifying hemagglutinin content by single radial immunodiffusion (SRID) gave indistinct or indeterminate zone margins for the RESVIR 9 component of commercial influenza vaccines. Initial studies suggested that the indeterminate zone may be related to interactions of detergents in production or in performance of the SRID. When the currently used zwitterionic detergent was used alone, the edge of the SRID zone of some vaccines was poorly defined; however, addition of a non-ionic detergent, Triton x-100, resulted in well defined zone margins. Vaccines manufactured with Triton x-100 and similar detergents gave consistently well defined zone margins. The observation needs further investigation to determine whether the current detergent for SRID should be changed or individualized for specific hemagglutinins. We initiated additional laboratory studies to improve current methods for producing reassortant influenza viruses for use as reference strains and for studies of host adaptation and virulence. High yield reassortants of influenza B viruses have not previously been available. However, we selected strains from the two currently circulating influenza B lineages, and produced strains that appear to be reassortants with high yield characteristics. In addition, we have begun to manipulate viruses by the method termed reverse genetics. We developed a clone of the matrix gene of PR8 to use for producing high yield strains and to provide a tool for further investigations of the features of the gene and its products that increase viral yield. These studies continue in order to improve understanding of the biology of influenza viruses and the factors affecting influenza virus replication.